Process for Producing Polypeptides

ABSTRACT

A process is described for producing a polypeptide heterologous to  E. coli  wherein  E. coli  cells comprising nucleic acid encoding the polypeptide are cultured in a culture medium while feeding to the culture medium a transportable organophosphate, such that the nucleic acid is expressed. The polypeptide is then recovered from the cells.

RELATED APPLICATIONS

This application is a continuation application of Ser. No. 11/077,272filed on Mar. 10, 2005, which application claims priority to U.S.Provisional Application No.: 60/552,678 filed Mar. 11, 2004, thecontents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a process for producing a polypeptideheterologous to E. coli. More particularly, the invention is directed tousing organophosphate to improve yield of such polypeptides.

2. Description of Related Art

Expression of heterologous proteins by Escherichia coli aided by thewell-understood molecular biology and relative ease in geneticmanipulation of the microorganism, has been very productive in bothlaboratory and industry. Typically, an inducible promoter (for example,the alkaline phosphatase promoter, the tac promoter, the arabinosepromoter, etc.) is employed for the regulation of heterologous proteinexpression. The requirement of an induction event provides theresearcher the opportunity to manage the timing of expression of thetarget protein. This ability is especially important for thoseheterologous proteins that are not well tolerated at high concentrationsby the host. By achieving desirable cell density prior to the inductionof expression, the volumetric yield of the desired protein may bemaximized.

Cells cease to grow when the microorganism is deprived of a requirednutrient. The limiting component may be carbon, nitrogen, phosphate,oxygen or any of the elements required by the cell. Under suchconditions, the cells exit from the growth phase. A way to alleviate theculture of the stress responses caused by the nutrient limitation is toprovide a feed of the lacking component. Common feeds introduced intofed-batch fermentation processes include glucose, amino acids, oxygen,etc.

In the case of cellular phosphorus (P), the requirement for phosphatesupply is not surprising given that P is the fifth most abundant elementin a cell behind carbon, oxygen, nitrogen, and hydrogen. Slanier,Adelberg and Ingraham, The Microbial World, 4^(th) ed. (Prentice Hall,NJ 1976), p. 1357. Phosphorus is an essential component in numerousmacromolecules such as nucleic acids, liposaccharides and membranelipids. Furthermore, its role in the high-energy phosphoanhydride bondsmakes it especially important in energy metabolism. E. coli is capableof utilizing inorganic phosphate (Pi), organophosphate or phosphonate asthe primary P source. The uptake of Pi from the environment can beachieved through two transporter systems, the Pit and the Pst systems.For the organophosphates, most are non-transportable and they first needto be hydrolyzed enzymatically in the periplasm before the released Pican be taken up by the Pi transport system(s). Only a feworganophosphates are transportable, and glycerol-3-phosphate (G3P) isone such example. G3P and glycerophosphate-1-phosphate (GIP) are knownas alpha-glycerophosphates. In response to Pi-limitation andcarbon-limitation, E. coli is capable of taking up available intact G3Pfrom the external environment into the intracellular compartment, whereG3P is metabolized to yield needed phosphate or carbon. Wanner,“Phosphorus Assimulation and Control of the Phosphate Regulon”, inEscherichia coli and Salmonella Cellular and Molecular Biology,Neidhardt, ed., (second edition), American Society for MicrobiologyPress (1996), pp. 1357-1365.

Further references on G3P are Silhavy et al, J. Bacteriol., 126: 951-958(1976) on the periplasmic protein related to the sn-glycerol-3-phosphatetransport system of E. coli; Argast et al., J. Bacteriol., 136:1070-1083 (1978) on a second transport system forsn-glycerol-3-phosphate in E. coli; Elvin et al., J. Bacteriol., 161:1054-1058 (1985) on Pi exchange mediated by the glpT-dependent G3Ptransport system; Rao et al., J. Bacteriol., 175: 74-79 (1993) on theeffect of glpT and glpD mutations on expression of the phoA gene in E.coli; and Elashvili et al., Appl. Environ. Microbiol., 64: 2601-2608(1998) on phnE and glpT genes enhancing utilization of organophosphatesin E. coli K-12. Further, Vergeles et al., Eur. J. Biochem., 233:442-447 (1995) disclose the high efficiency of glycerol-2-phosphate(G2P), otherwise known as beta-glycerophosphate, and G3P as nucleotidylacceptors in snake venom phosphodiesterase esterifications.

The current understanding of the two transport systems for the uptake ofexogenous G3P in E. coli, the Ugp and GlpT transport systems, has beenwell summarized in the book Escherichia coli and Salmonella, Cellularand Molecular Biology edited by Neidhardt et. al. (second edition),supra, pp. 1364 referring to references 13 and 81. The Ugp operonbelongs to the pho regulon. It is induced by phosphate limitation andpositively regulated by phoB protein. The Ugp system is a periplasmicbinding protein-dependent multi-component transport system, with ugpBencoding the periplasmic binding protein, ugpA and ugpC encodingintegral membrane channel proteins, and ugpC encoding ATPase. GlpT ispart of the glp system that mediates the uptake and metabolism ofglycerol, G3P, and glycerol phosphoryl phosphodiesters (Lin et al.,Annu. Rev. Microbiol., 30: 535-578 (1976); Chapter 20; pg 307-342Dissimilatory Pathways for sugars, polyols and carboxylates. Escherichiacoli and Salmonella, Cellular and Molecular Biology, second edition).This transport system is an anion exchanger that is known to mediate theefflux of Pi from the cytoplasm by exchange with external G3P. In awild-type strain growing on G3P, while little Pi is released by cellstaking up G3P via the Ugp system, Pi can be released into the periplasmwhen G3P is taken up via the GlpT system. If a repressive amount of Piis released as a result of glpT-permease-mediated efflux, the phoregulon activity, the Ugp system included, will be shut off. Undercertain conditions, GlpT is the only route for the exit of Pi from thecell by exchange with external G3P. Elvin et al., J. Bacteriol., 161:1054-1058 (1985); Rosenberg, “Phosphate transport in prokaryotes,” p.205-248. In B. P. Rosen and S. Silver (ed.), Ion Transport inProkaryotes (Academic Press, Inc., New York, 1987).

When the capacities of the Ugp and the GlpT systems are compared totransport G3P, the maximal velocities of the two systems are similar.The apparent affinity for G3P is higher with the Ugp system than withthe GlpT system. Likely, both systems will be able to supply enough G3Pfor cell growth if available in the growth medium. However, G3Ptransported exclusively via the Ugp system can serve as the sole sourceonly of phosphate but not of carbon, while GlpT-transported G3P canserve as the sole source for both (Schweizer et al., J. Bacteriol., 150:1154-1163 (1982)). The two ugp genes coding for thepho-regulon-dependent G3P transport system have been mapped (Schweizeret al., J. Bacteriol., 150: 1164-1171 (1982)), the ugp region containingthese genes has been characterized (Schweizer et al., Mol. and Gen.Genetics, 197: 161-168 (1984)), and the regulation of ugp operon studied(Schweizer et al., J. Bacteriol., 163: 392-394 (1985); Kasahara et al.,J. Bacteriol., 173: 549-558 (1991); Su et al., Molecular & GeneralGenetics, 230: 28-32 (1991); Brzoska et al., “ugp-dependent transportsystem for sn-glycerol 3-phosphate of Escherichia coli,” p. 170-177 inA. Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E. Yagil(ed.), Phosphate Metabolism and Cellular Regulation in Microorganisms(American Society for Microbiology, Washington, D.C., 1987); Brzoska etal., J. Bacteriol., 176: 15-20 (1994); and Xavier et al., J. Bacteriol.,177: 699-704 (1995)).

In wild-type strains, there exists a stable intracellular pool of G3Pand it is maintained at approximately 200 μM. Internally, G3P can besynthesized by the enzymatic conversion of glycerol by glycerol kinase(encoded by glpK) to G3P when grown on glycerol as the sole carbonsource, or from the reduction of the glycolytic intermediate,dihydroxyactone phosphate, by G3P synthase, the gene product of the gpsAgene, during growth on carbon sources other than glycerol. Since G3P isan important intermediate that forms the scaffold of all phospholipidmolecules, internal glycerol phosphates may also be generated from thebreakdown of phospholipids and triacylglycerol. As a metabolite,internal G3P may be. channeled into the phospholipid biosyntheticpathway or be oxidized by G3P dehydrogenase to form dihydroxyacetonephosphate and fed into the glycolytic pathway.

In situations where the AP promoter is employed for regulatingheterologous protein expression in E. coli, since induction occurs onlyafter the medium is depleted of Pi, cells induced for AP promoteractivity are typically starved for phosphate and in a declining state ofhealth. They may have to scavenge for phosphate needed for cellularfunctions. Possible consequences of such phosphate scavenging mayinclude turnover of ribosomes, lower cell energetics, and increasedprotease expression and proteolysis (St. John and Goldberg, J.Bacteriol., 143: 1223-1233 (1980)), potentially leading to less healthycells with reduced capacity for protein accumulation.

Improving the metabolic state of E. coli may conceivably increase thecapacity of the cell to synthesize proteins. If phosphate is fed slowly,the cells may only sense low Pi concentration in the periplasm, therebyinducing the pho regulon without being starved intracellularly for the Patom (see U.S. Pat. No. 5,304,472). There is a need for providingfurther methods of producing heterologous polypeptides in E. coli.

SUMMARY OF THE INVENTION

In the invention herein, a process is provided for improving theexpression of heterologous polypeptides in E. coli. The feeding oftransportable organophosphate such as an alpha-glycerophosphate tovarious E. coli hosts, including those with and without the wild-typeglpT gene and those with and without the wild-type phoA gene, such as,for example, (ugp+ΔglpT phoA−) E. coli, is shown to improve theexpression of heterologous protein at both shake-flask and10-L-fermentor scale, and is expected to perform similarly at largerscale such as 10,000L. Product yield benefit was observed acrossmultiple model systems that employed a variety of promoters, includinginducible promoters such as the tac, T7 or AP promoter, for theexpression of the heterologous proteins. A further advantage is that theproduct can be obtained earlier in the active growth phase, i.e., in ashorter time than otherwise. In certain embodiments, more product can beobtained earlier in the active growth phase to improve productivitysignificantly.

Accordingly, the present invention is as claimed. In one aspect thepresent invention provides a process for producing a polypeptideheterologous to E. coli comprising (a) culturing E. coli cellscomprising nucleic acid encoding the polypeptide in a culture mediumwhile feeding to the culture medium a transportable organophosphate,such that the nucleic acid is expressed, and (b) recovering thepolypeptide from the cells. In a preferred embodiment, theorganophosphate is a glycerophosphate, more preferably, analpha-glycerophosphate and/or a beta-glycerophosphate , and still morepreferably, a mixture of glycerol-2-phosphate and glycerol-3-phosphateor glycerol-3-phosphate alone. In another preferred aspect, theculturing takes place in a shake flask or fermentor, preferably afermentor. In yet another preferred embodiment, the polypeptide isrecovered from the cytoplasm, periplasm, or culture medium of the cells.Also preferred is that expression of the nucleic acid is regulated by aninducible promoter, such as alkaline phosphatase promoter, tac promoter,or T7 promoter, and preferably wherein expression of the nucleic acidbegins while in the active growth phase of the culturing step. In oneembodiment, the E. coli is wild type. In another embodiment, the E. coliis deficient in chromosomal glpT and in chromosomal phoA, but preferablynot deficient in chromosomal ugp. Preferably an inorganic phosphate isalso present during the culturing step.

Without being limited to any one theory, it is believed that in thisprocess the transportable organophosphate compounds are fed to the cellssuch that the phosphate supply will not be sensed by the pstS of the Phosystem but will still provide phosphate upon breakdown in the cytoplasm,and further that feeding transportable organophosphate such as G3Ppotentially enriches the cells with a utilizable metabolic intermediatethat can be readily fed into important metabolic pathways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows expression of a secreted llama antibody fragment in a BL21E. coli host using the tac promoter in a shake-flask culture, utilizingeither water or 200 mM G3P as a supplement in low-phosphate (CRAP) orhigh-phosphate (THCD) medium.

FIG. 2 shows expression of a cytoplasmic Apo2L in a HMS174 E. coli hostusing the T7 promoter in a shake-flask culture, utilizing either wateror 200 mM G3P as a supplement in CRAP medium.

FIG. 3 shows the effect of feeding of G3P during fermentation onsecreted IGF-1 accumulation over time. This uses a wild-type E. colihost, the AP promoter, and continuously fed glucose.

FIG. 4 shows the effect of a glpT mutation and G3P feeding duringfermentation on secreted IGF-1 accumulation over time. This uses a ΔglpTE. coli host, the AP promoter, and varying G3P feed rate.

FIG. 5 shows the plasmid diagram for pAPApo2-P2RU.

FIG. 6 shows the nucleotide sequence of human Apo-2 ligand cDNA (SEQ IDNO:1) and its derived amino acid sequence (SEQ ID NO:2). The “N” atnucleotide position 447 (in SEQ ID NO:1) is used to indicate thenucleotide base may be a “T” or “G”.

FIG. 7 shows the effect of G3P feeding on specific accumulation of Apo2Lin the ΔglpT E. coli (43F6) host, with three different feed rates and acontrol with no G3P feed.

FIG. 8 shows the benefit on the specific total accumulation of Apo2L offeeding glycerophosphate over inorganic phosphate to the wild-type glpThost (43E7), wherein the cell density increases to over 200 OD550.

FIG. 9 shows the effect on specific total accumulation of Apo2L ofreplacement of inorganic phosphate with glycerophosphate in thewild-type glpT E. coli host (43E7) and ΔglpT E. coli (43F6) host.

FIG. 10 shows the effect on total Apo2L accumulation of replacement ofalpha-glycerophosphate with a 50:50 mixture of alpha- andbeta-glycerophosphate as a feed, versus a no-feed control, in a ΔglpT E.coli (61G1) host.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Definitions

As used herein, “polypeptide” refers generally to peptides and proteinshaving more than about ten amino acids. “Heterologous” polypeptides arethose polypeptides foreign to the host cell being utilized, such as ahuman protein produced by E. coli. While the polypeptide may beprokaryotic or eukaryotic, preferably it is eukaryotic, more preferablymammalian, and most preferably human.

Examples of mammalian polypeptides include molecules such as, e.g.,rennin; a growth hormone, including human growth hormone or bovinegrowth hormone; growth-hormone releasing factor; parathyroid hormone;thyroid-stimulating hormone; lipoproteins; 1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; thrombopoietin;follicle-stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnaturietic factor; lung surfactant; a plasminogen activator, such asurokinase or human urine or tissue-type plasminogen activator (t-PA);bombesin; thrombin; hemopoietic growth factor; tumor necrosisfactor-alpha and -beta; antibodies to ErbB2 domain(s) such as 2C4 (WO01/00245; hybridoma ATCC HB-12697), which binds to a region in theextracellular domain of ErbB2 (e.g., any one or more residues in theregion from about residue 22 to about residue 584 of ErbB2, inclusive);enkephalinase; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors; integrin; protein A or D; rheumatoid factors; a neurotrophicfactor such as brain-derived neurotrophic factor (BDNF), neurotrophin-3,-4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor suchas NGF; cardiotrophins (cardiac hypertrophy factor) such ascardiotrophin-1 (CT-1); platelet-derived growth factor (PDGF);fibroblast growth factor such as aFGF and bFGF; epidermal growth factor(EGF); transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF- 1, TGF- 2, TGF- 3, TGF- 4, or TGF- 5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I);insulin-like growth factor binding proteins; CD proteins such as CD-3,CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); an interferon such asinterferon-alpha, -beta, and -gamma; a serum albumin, such as humanserum albumin (HSA) or bovine serum albumin (BSA); colony stimulatingfactors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs),e.g., IL-1 to IL-10; anti-HER-2 antibody; Apo2 ligand (Apo2L);superoxide dismutase; T-cell receptors; surface-membrane proteins;decay-accelerating factor; viral antigens such as, for example, aportion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; antibodies; and fragments of any of theabove-listed polypeptides.

The preferred polypeptides of interest include polypeptides such as HSA,BSA, anti-IgE, anti-CD20, anti-IgG, t-PA, gp120, anti-CD11a, anti-CD18,2C4, anti-VEGF, VEGF, TGF-beta, activin, inhibin, anti-HER-2, DNase,IGF-I, IGF-II, brain IGF-I, growth hormone, relaxin chains,growth-hormone releasing factor, insulin chains or pro-insulin,antibodies and antibody fragments, NGF, NT-3, BDNF, Apo2L, andurokinase. The polypeptide is most preferably IGF-I or Apo2L.

The terms “Apo2 ligand,” “Apo2L,” and “TRAIL” are used hereininterchangeably to refer to a polypeptide sequence that includes aminoacid residues 114-281, inclusive, residues 95-281, inclusive, residues92-281, inclusive, residues 91-281, inclusive, residues 41-281,inclusive, residues 15-281, inclusive, or residues 1-281, inclusive, ofthe amino acid sequence shown in FIG. 6 (SEQ ID NO:2), as well asbiologically active fragments, and deletional, insertional, orsubstitutional variants of the above sequences. In one embodiment, thepolypeptide sequence comprises residues 114-281 of FIG. 6 (SEQ ID NO:2).Optionally, the polypeptide sequence comprises residues 92-281 orresidues 91-281 of FIG. 6 (SEQ ID NO:2). The Apo2L polypeptides may beencoded by the native nucleotide sequence shown in FIG. 6 (SEQ ID NO:1). Optionally, the codon that encodes residue Pro119 (FIG. 6; SEQ IDNO:1) may be “CCT” or “CCG.” In another preferred embodiment, thefragments or variants are biologically active and have at least about80% amino acid sequence identity, more preferably at least about 90%sequence identity, and even more preferably, at least 95%, 96%, 97%,98%, or 99% sequence identity, with any one of the above sequences. Thedefinition encompasses substitutional variants of Apo2 ligand in whichat least one of its native amino acids is substituted by an alanineresidue. The definition also encompasses a native-sequence Apo2 ligandisolated from an Apo2 ligand source or prepared by recombinant orsynthetic methods. The Apo2 ligand of the invention includes thepolypeptides referred to as Apo2 ligand or TRAIL disclosed in WO97/01633, WO 97/25428, and WO 01/00832. The terms “Apo2 ligand” and“Apo2L” are used to refer generally to forms of the Apo2 ligand thatinclude monomer, dimer, or trimer forms of the polypeptide. Allnumbering of amino acid residues referred to in the Apo2L sequence usesthe numbering according to FIG. 6 (SEQ ID NO:2) unless specificallystated otherwise. For instance, “D203” or “Asp203” refers to theaspartic acid residue at position 203 in the sequence provided in FIG. 6(SEQ ID NO:2).

The term “Apo-2 ligand extracellular domain” or “Apo2 ligand ECD” refersto a form of Apo2 ligand that is essentially free of transmembrane andcytoplasmic domains. Ordinarily, the ECD will have less than 1% of suchtransmembrane and cytoplasmic domains, and preferably will have lessthan 0.5% of such domains. “Biologically active” or “biologicalactivity,” as it relates to Apo2L, refers to (a) having the ability toinduce or stimulate apoptosis in at least one type of mammalian cancercell or virally infected cell in vivo or ex vivo; (b) capable of raisingan antibody (i.e., immunogenic), (c) capable of binding and/orstimulating a receptor for Apo2L; or (d) retaining the activity of anative or naturally occurring Apo2L polypeptide.

The expression “control sequences” refers to DNA sequences necessary forthe expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotesinclude a promoter, optionally an operator sequence, and aribosome-binding site.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a pre-protein that participates in thesecretion of the polypeptide; a promoter is operably linked to a codingsequence if it affects the transcription of the sequence; or aribosome-binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. Linking isaccomplished by ligation at convenient restriction sites. If such sitesdo not exist, the synthetic oligonucleotide adaptors or linkers may beused in accordance with conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that all progeny may notbe precisely identical in DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “organophosphate” as used herein refers to a phosphate compoundcontaining one or more carbon atoms, which can also contain halideatoms. Such phosphate compound must be such that it can be fed to andutilized by a cell culture. These compounds are often used aspesticides. “Transportable” organophosphates can be transported from theexternal environment of the cell into the cell without having to bepre-hydrolyzed in any way. If an E. coli strain does not grow well withan organophosphate, the utilization of such organophosphate can beenhanced by overexpressing in E. coli the phnE gene product. Such geneconfers the spontaneous organophosphate utilization phenotype to the E.coli strain upon transformation. See Elashvili et al., supra. Examplesof suitable organophosphates include alkyl halophosphates such asdiisopropyl fluorophosphate, alkyl phosphates such as diisopropylphosphate and 3,4-dihydroxybutyl-1-phosphate, as well as sugar- oralkanol-containing phosphates such as hexose-6-phosphate andglycerol-3-phosphate. Glucose-1-phosphate, hexose-6-phosphate andglycerophosphates such as glucose-1-glycerophosphate,fructose-6-glycerophosphate, alpha-glycerophosphates such asglycerol-1-phosphate and glycerol-3-phosphate, and beta-glycerophosphate(glycerol-2-phosphate) are preferred, with glycerophosphates morepreferred, alpha- and/or beta-glycerophosphates still more preferred,and glycerol-2-phosphate and/or glycerol-3-phosphate still morepreferred, and a mixture of glycerol-2- and glycerol-3-phosphate orglycerol-3-phosphate most particularly preferred herein for use. As usedherein, the term “G3P” without being in a mixture or “G3P alone” refersto a composition containing at least about 80% glycerol-3-phosphate; itmay contain up to about 20% impurities such as G2P. A mixture of G3P andG2P would contain less than about 80% G3P.

An inorganic phosphate is a phosphate compound that does not contain anycarbon atoms, with the phosphate typically being associated with analkali or alkali earth metal such as potassium, calcium, magnesium, orsodium phosphate.

“Active growth phase” refers to the phase of the culturing step whereinthe cells are actively growing and not severely nutrient-limited cellssuch as those that are in stationary phase.

Modes for Carrying Out the Invention

The present invention provides a method for producing polypeptidesheterologous to E. coli. In this method E. coli cells comprising nucleicacid encoding the polypeptide are cultured in a culture medium whilefeeding to the culture medium a transportable organophosphate, such thatthe nucleic acid is expressed. The polypeptide is then recovered fromthe cells. The recovery may be from the cytoplasm, periplasm, or culturemedium of the cells. The culturing may take place in any suitablevessel, preferably a shake flask or fermentor, more preferably, in afermentor.

Culturing parameters are used and polypeptide production may beconducted in a conventional manner, such as those procedures describedbelow.

-   A. Selection of Nucleic Acid and Modifications Thereof

The nucleic acid encoding the polypeptide of interest is suitably RNA,cDNA, or genomic DNA from any source, provided it encodes thepolypeptide(s) of interest. Methods are well known for selecting theappropriate nucleic acid for expression of heterologous polypeptides(including variants thereof) in E. coli.

If monoclonal antibodies are being produced, DNA encoding the monoclonalantibodies is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains ofmurine antibodies). The hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transformed into the bacterial host cells herein toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5: 256-262 (1993) and Plückthun, Immunol. Revs., 130: 151-188 (1992).

Methods for humanizing non-human antibodies have been described in theart. Preferably, a humanized antibody has one or more amino acidresidues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the method of Winterand co-workers (Jones et al., Nature, 321: 522-525 (1986); Riechmann etal., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting hypervariable region sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567)wherein substantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901(1987)). Another method uses a particular framework region derived fromthe consensus sequence of all human antibodies of a particular subgroupof light or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablethat illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general., the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

Various forms of the humanized antibody or affinity-matured antibody arecontemplated. For example, the humanized antibody or affinity-maturedantibody may be an antibody fragment, such as a Fab, that is optionallyconjugated with one or more targeting agent(s) in order to generate animmunoconjugate. Alternatively, the humanized antibody oraffinity-matured antibody may be an intact antibody, such as an intactIgG1 antibody.

Fab′-SH fragments can be directly recovered from E. coli and chemicallycoupled to form F(ab′)₂ fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). According to another approach, F(ab′)₂ fragments can beisolated directly from recombinant host cell culture. Other techniquesfor the production of antibody fragments will be apparent to the skilledpractitioner. In other embodiments, the antibody of choice is asingle-chain Fv fragment (scFv) (WO 93/16185; U.S. Pat. Nos. 5,571,894and 5,587,458). The antibody fragment may also be a “linear antibody”,e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibodyfragments may be monospecific or bispecific.

Bispecific antibodies are antibodies that have binding specificities forat least two different epitopes. Exemplary bispecific antibodies maybind to two different epitopes of the same protein. Bispecificantibodies can be prepared as full-length antibodies or antibodyfragments (e.g., F(ab′)₂ bispecific antibodies). These may be as fusionsof various antibody chains or can be one chain. One heavy chain can becompetent by itself.

In one approach to producing bispecific antibodies, a bispecificimmunoadhesin is prepared by introducing into a host cell DNA sequencesencoding a first fusion comprising a first binding domain fused to animmunoglobulin heavy-chain constant domain sequence lacking alight-chain binding site; a second fusion comprising a second bindingdomain fused to an immunoglobulin heavy-chain constant domain sequenceretaining a light-chain binding site; and an immunoglobulin light-chain,respectively. The host cells are then cultured so as to express the DNAsequences to produce a mixture of (i) a heterotrimer comprising thefirst fusion covalently linked with a second fusion-immunoglobulinlight-chain pair; (ii) a heterotetramer comprising two covalently linkedsecond fusion-immunoglobulin light-chain pairs; and (iii) a homodimercomprising two covalently linked molecules of the first fusion. Themixture of products is removed from the cell culture and theheterotrimer is isolated from the other products. This approach isdisclosed in WO 94/04690. For further details of generating bispecificantibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

According to another approach described in U.S. Pat. No. 5,731,168, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed, for example, inU.S. Pat. No. 4,676,980, along with a number of cross-linkingtechniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)₂ fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Additionally, Fab′-SH fragments can be directly recovered from E. coliand chemically coupled to form bispecific antibodies (Shalaby et al., J.Exp. Med., 175: 217-225 (1992)).

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers (Kostelny et al., J. Immunol., 148: 1547-1553 (1992)).The leucine zipper peptides from the Fos and Jun proteins are linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers are reduced at the hinge region to form monomers andthen re-oxidized to form the antibody heterodimers. This method can alsobe utilized for the production of antibody homodimers. The “diabody”technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA,90: 6444-6448 (1993) has provided an alternative mechanism for makingbispecific antibody fragments. The fragments comprise a heavy-chainvariable domain (V_(H)) connected to a light-chain variable domain(V_(L)) by a linker that is too short to allow pairing between the twodomains on the same chain. Accordingly, the V_(H) and V_(L) domains ofone fragment are forced to pair with the complementary V_(L) and V_(H)domains of another fragment, thereby forming two antigen-binding sites.Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported (Gruber et al., J.Immunol., 152: 5368 (1994)).

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared (Tutt et al., J. Immunol., 147:60 (1991)).

Nucleic acid molecules encoding polypeptide variants are prepared by avariety of methods known in the art. These methods include, but are notlimited to, isolation from a natural source (in the case of naturallyoccurring amino acid sequence variants) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, or cassette mutagenesis of an earlier prepared variant or anon-variant version of the polypeptide.

It may be desirable to modify the antibody of the invention with respectto effector function, e.g., so as to enhance Fe receptor binding. Thismay be achieved by introducing one or more amino acid substitutions intoan Fc region of the antibody. Alternatively or additionally, cysteineresidue(s) may be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region.

To increase the serum half-life of the antibody, one may incorporate asalvage receptor binding epitope into the antibody (especially anantibody fragment) as described in U.S. Pat. No. 5,739,277, for example.As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Other modifications of the antibody are contemplated herein. Forexample, the antibody may be linked to one of a variety ofnon-proteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol.

-   B. Insertion of Nucleic Acid Into a Replicable Vector

The heterologous nucleic acid (e.g., cDNA or genomic DNA) is suitablyinserted into a replicable vector for expression in the E. coli underthe control of a suitable promoter. Many vectors are available for thispurpose, and selection of the appropriate vector will depend mainly onthe size of the nucleic acid to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components depending on the particular host cell withwhich it is compatible. Depending on the particular type of host, thevector components generally include, but are not limited to, one or moreof the following: a signal sequence, an origin of replication, one ormore marker genes, a promoter, and a transcription termination sequence.

In general, plasmid vectors containing replicon and control sequencesthat are derived from species compatible with the host cell are used inconnection with E. coli hosts. The vector ordinarily carries areplication site, as well as marking sequences that are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (see, e.g., Bolivar et al., Gene, 2: 95 (1977)). pBR322contains genes for ampicillin and tetracycline resistance and thusprovides easy means for identifying transformed cells. The pBR322plasmid, or other bacterial plasmid or phage, also generally contains,or is modified to contain, promoters that can be used by the E. colihost for expression of the selectable marker genes.

(i) Signal Sequence Component

The DNA encoding the polypeptide of interest herein may be expressed notonly directly, but also as a fusion with another polypeptide, preferablya signal sequence or other polypeptide having a specific cleavage siteat the N-terminus of the mature polypeptide. In general, the signalsequence may be a component of the vector, or it may be a part of thepolypeptide-encoding DNA that is inserted into the vector. Theheterologous signal sequence selected should be one that is recognizedand processed (i.e., cleaved by a signal peptidase) by the host cell.

For prokaryotic host cells that do not recognize and process the nativeor a eukaryotic polypeptide signal sequence, the signal sequence issubstituted by a prokaryotic signal sequence, selected, for example,from the group consisting of the alkaline phosphatase, penicillinase,Ipp, or heat-stable enterotoxin II leaders.

(ii) Origin of Replication Component

Expression vectors contain a nucleic acid sequence that enables thevector to replicate in one or more selected host cells. Such sequencesare well known for a variety of bacteria. The origin of replication fromthe plasmid pBR322 is suitable for most Gram-negative bacteria such asE. coli.

(iii) Selection Gene Component

Expression vectors generally contain a selection gene, also termed aselectable marker. This gene encodes a protein necessary for thesurvival or growth of transformed host cells grown in a selectiveculture medium. Host cells not transformed with the vector containingthe selection gene will not survive in the culture medium. Thisselectable marker is separate from the genetic markers as utilized anddefined by this invention. Typical selection genes encode proteins that(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies other than those caused by the presence of the geneticmarker(s), or (c) supply critical nutrients not available from complexmedia, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. In this case, those cells that are successfully transformedwith the nucleic acid of interest produce a polypeptide conferring drugresistance and thus survive the selection regimen. Examples of suchdominant selection use the drugs neomycin (Southern et al., J. Molec.Appl. Genet., 1: 327 (1982)), mycophenolic acid (Mulligan et al.,Science, 209: 1422 (1980)), or hygromycin (Sugden et al., Mol. Cell.Biol., 5: 410-413 (1985)). The three examples given above employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid),or hygromycin, respectively.

(iv) Promoter Component

The expression vector for producing the polypeptide of interest containsa suitable promoter that is recognized by E. coli and is operably linkedto the nucleic acid encoding the polypeptide of interest. Promoterssuitable for use with E. coli hosts include the beta-lactamase andlactose promoter systems (Chang et al., Nature, 275: 615 (1978); Goeddelet al., Nature, 281: 544 (1979)), the arabinose promoter system (Guzmanet al., J. Bacteriol., 174: 7716-7728 (1992)), alkaline phosphatase, theT7 promoter, a tryptophan (trp) promoter system (Goeddel, Nucleic AcidsRes., 8: 4057 (1980) and EP 36,776) and hybrid promoters such as the tacpromoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)).However, other known bacterial promoters are suitable. Their nucleotidesequences have been published, thereby enabling a skilled workeroperably to ligate them to DNA encoding the polypeptide of interest(Siebenlist et al., Cell, 20: 269 (1980)) using linkers or adaptors tosupply any required restriction sites.

Preferably, the promoter employed herein is an inducible promoter, i.e.,one that is activated by an inducing agent or condition (such asperiplasmic phosphate depletion). Preferred such inducible promotersherein are the alkaline phosphatase promoter, the tac promoter, or theT7 promoter.

Promoters for use in bacterial systems also generally contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding thepolypeptide of interest. The promoter can be removed from the bacterialsource DNA by restriction enzyme digestion and inserted into the vectorcontaining the desired DNA.

(v) Construction and Analysis of Vectors

Construction of suitable vectors containing one or more of theabove-listed components employs standard ligation techniques. Isolatedplasmids or DNA fragments are cleaved, tailored, and re-ligated in theform desired to generate the plasmids required.

For analysis to confirm correct sequences in plasmids constructed, theligation mixtures are used to transform E. coli K12 strain 294 (ATCC31,446) or other strains, and successful transformants are selected byampicillin or tetracycline resistance where appropriate. Plasmids fromthe transformants are prepared, analyzed by restriction endonucleasedigestion, and/or sequenced by the method of Sanger et al., Proc. Natl.Acad. Sci. USA, 74: 5463-5467 (1977) or Messing et al., Nucleic AcidsRes., 9: 309 (1981), or by the method of Maxam et al., Methods inEnzymology, 65: 499 (1980).

-   C. Selection and Transformation of Host Cells

E. coli hosts suitable as parental hosts for expression plasmids hereininclude E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E. coliB, and E. coli X1776 (ATCC 31,537). These examples are illustrativerather than limiting. Mutant cells of any of the above-mentioned strainsmay also be employed as the starting hosts that are then further mutatedto contain at least the minimum genotype required herein. E. coli strainW3110 is a preferred parental host because it is a common host strainfor recombinant DNA product fermentations. Examples of starting E. colihosts to be used as parent hosts, along with their genotypes, areincluded in the table below: Strain Genotype W3110 K-12F⁻lambda⁻IN(rrnD-rrnE)1 1A2 ΔfhuA (ΔtonA) 9E4 ΔfhuA (ΔtonA) ptr3 27A7ΔfhuA (ΔtonA) ptr3 phoAΔE15 (argF-lac)169 27C6 ΔfhuA (ΔtonA) phoAΔE15(argF-lac)169 ptr3 ompT Δ(nmpc-fepE) 27C7 ΔfhuA (ΔtonA) phoAΔE15(argF-lac)169 ptr3 degP41::kanR ompT Δ(nmpc-fepE) 33D3 ΔfhuA (ΔtonA)ptr3 lacIq lacL8 ompT Δ(nmpc- fepE) degP::kanR 36F8 ΔfhuA (ΔtonA)phoAΔE15 (argF-lac)169 ptr3 degP41::kanR ilvG+ 43D3 ΔfhuA (ΔtonA)phoAΔE15 (argF-lac)169 ptr3 degP41::kanR ompT Δ(nmpc-fepE) ilvG+ 43E7ΔfhuA (ΔtonA) phoAΔE15 (argF-lac)169 ptr3 degP41 ompT Δ(nmpc-fepE) ilvG+43F6 ΔfhuA (ΔtonA) phoAΔE15 (argF-lac)169 ptr3 degP41::kanR ompTΔ(nmpc-fepE) Δ(rbs7) ilvG+ ΔglpT596 44D6 ΔfhuA (ΔtonA) (argF-lac)169ptr3 degP41::kanR ompT Δ(nmpc-fepE) ilvG+ 45F8 ΔfhuA (ΔtonA)(argF-lac)169 ptr3 degP41 ompT Δ(nmpc-fepE) ilvG+ phoS(T10Y) 45F9 ΔfhuA(ΔtonA) (argF-lac)169 ptr3 degP41 ompT Δ(nmpc-fepE) ilvG+ phoS(T10Y)cyo::kanR 61G1 ΔfhuA Δptr ΔompT ΔdegP ΔphoA ilvG+ ΔglpTQ

Also suitable are the intermediates in making strain 36F8, i.e., 27B4(U.S. Pat. No. 5,304,472) and 35E7 (a spontaneous temperature-resistantcolony isolate growing better than 27B4). An additional suitable strainis the E. coli strain having the mutant periplasmic protease(s)disclosed in U.S. Pat. No. 4,946,783 issued Aug. 7, 1990.

In one embodiment, the E. coli host cell employed is wild type withrespect to or in reference to the glpT gene, such as 43E7, or isdeficient in the glpT gene, such as 43F6 or 61G1. In another embodiment,the E. coli host cell employed is wild type with respect to or inreference to the phoA gene. In a preferred embodiment, the E. coli isdeficient in chromosomal phoA. In another preferred embodiment, the E.coli is deficient in chromosomal glpT and in chromosomal phoA. In a morepreferred embodiment, the E. coli is deficient in chromosomal glpT andin chromosomal phoA, but not in chromosomal ugp. The most preferred suchmutant E. coli host is 43F6 or 61G1, the genotypes of which are given inthe above table. As used herein, “wild type with respect to glpT” refersto E. coli hosts that are glpT+ or glpT competent cells, i.e., thosethat are not deficient in chromosomal glpT. Similarly, as used herein,“wild type with respect to phoA” refers to E. coli hosts that are phoA+or phoA competent cells, i.e., those that are not deficient inchromosomal phoA.

The strains of this invention may be produced by chromosomal integrationof the parental strain or other techniques, including those set forth inthe Examples below.

The nucleic acid encoding the polypeptide is inserted into the hostcells. Preferably, this is accomplished by transforming the host cellswith the above-described expression vectors and culturing inconventional nutrient media modified as appropriate for inducing thevarious promoters.

Transformation means introducing DNA into an organism so that the DNA isreplicable, either as an extrachromosomal element or by chromosomalintegrant. Depending on the host cell used, transformation is done usingstandard techniques appropriate to such cells. The calcium treatmentemploying calcium chloride, as described in section 1.82 of Sambrook etal., Molecular Cloning: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 1989), is generally used for prokaryotic cellsor other cells that contain substantial cell-wall barriers. Anothermethod for transformation employs polyethylene glycol/DMSO, as describedin Chung and Miller, Nucleic Acids Res., 16: 3580 (1988). Yet anothermethod is the use of the technique termed electroporation.

-   D. Culturing the Host Cells

E. coli cells used to produce the polypeptide of interest are culturedin suitable media as described generally in Sambrook et al., supra. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

The cells are cultured while the culture medium is fed with atransportable organophosphate such as a glycerophosphate, e.g.,alpha-glycerophosphate and/or beta-glycerophosphate, and especiallyglycerol-2-phosphate and/or glycerol-3-phosphate. The culturing may takeplace in a shake flask or a fermentor, preferably a fermentor. Thepolypeptide is preferably recovered from the cytoplasm, periplasm, orculture medium of the cells.

In the process of this invention, expression of the nucleic acid canbegin at any phase of the culturing step. However, preferably expressionof the nucleic acid begins while cell density is still increasing. Thiscan be accomplished by the inducement of the promoter with theappropriate inducer or inducing condition before cell growth ceases.

The feed rate of the organophosphate into the culture medium to beemployed for maximum production of the polypeptide depends on manyfactors, including the type of organophosphate, the concentration oforganophosphate, the type of polypeptide being produced, the type ofpromoter, the host cell strain employed, and the cell density in thebroth. If the polypeptide is IGF-1 and the organophosphate isglycerol-3-phosphate intended to extend the production duration, underthe culture conditions described and using a 10-L process, the feed rateof the organophosphate is preferably from about 1 to 7 mmoles/hour perabout 8-10 liters (see FIG. 4), more preferably from about 1 to 6mmoles/hour, and still more preferably from about 2 to 6 mmoles/hour,yet still more preferably from about 2 to 5 mmoles/hour, and mostpreferably from about 3 to 4 mmoles/hour. The optimal feed rate isdependent on the process, the cell density, the respiration rate, etc.

Also, in a preferred embodiment, where the polypeptide is Apo2L and theorganophosphate is glycerol-3-phosphate intending to shift productexpression to concur with the active growth phase and using a 10-Lprocess, the feed rate of the organophosphate is from about 4 to 17mmoles/hour per about 8-10 liters (see FIG. 7), more preferably fromabout 6 to 16 mmoles/hour, still more preferably from about 8 to 15mmoles/hour, and most preferably from about 10 to 14 mmoles/hour. Theoptimal feed rate of the organophosphate needs to be determined for theindividual process employed for the expression of the specificheterologous protein.

Any other necessary media ingredients besides carbon, nitrogen, andinorganic phosphate sources may also be included at appropriateconcentrations introduced alone or as a mixture with another ingredientor medium such as a complex nitrogen source. Preferably, an inorganicphosphate is also present in the culture medium at the start of theculturing step. If such inorganic phosphate, preferably sodium and/orpotassium phosphate, is present, the ratio of inorganic phosphate toorganophosphate depends on such factors as the type of polypeptideexpressed and organophosphate employed. This ratio can be anyproportion, as determined readily by those skilled in the art, rangingtypically from about 1:10 (one part of Pi to 10 parts oforganophosphate) to 1:0.25. For Apo2 ligand, preferably it ranges fromabout 1:4 to 1:0.25, and more preferably about 1:3 to 1:0.5, and yetmore preferably about 1:3 to 1:1, and still more preferably about 1:2 to1:1, and most preferably about 1:1. Such ratios allow earlier inductionof protein expression, and in some cases allow more product to beproduced earlier. The pH of the medium may be any pH from about 5-9,depending mainly on the host organism.

If the promoter is an inducible promoter, for induction to occur,typically the cells are cultured until a certain optical density isachieved, e.g., a A₅₅₀ of about 200 for a high-cell-density process, atwhich point induction is initiated (e.g., by addition of an inducer, bydepletion of a medium component, etc.), to induce expression of the geneencoding the polypeptide of interest.

Where the alkaline phosphatase promoter is employed, E. coli cells usedto produce the polypeptide of interest of this invention are cultured insuitable media in which the alkaline phosphatase promoter can be inducedas described generally, e.g., in Sambrook et al., supra. At first, themedium may contain inorganic phosphate for the growth of the bacteriumin an amount sufficiently large to support significant cell growth andavoid induction of synthesis of target heterologous polypeptide underthe promoter control. As the cells grow and utilize phosphate, theydecrease the level of inorganic phosphate in the medium, thereby causinginduction of synthesis of the polypeptide when the inorganic phosphateis exhausted. By adding, for example, a feed constituting a mixture ofG2P and G3P or a G3P feed, further growth to a higher cell density, suchas up to 200 OD550 or higher, takes place in the absence of inorganicphosphate or at starvation levels of inorganic phosphate in theperiplasm and supporting culture medium, resulting in an increase or anextension of product accumulation.

-   E. Detecting Expression

Gene expression may be measured in a sample directly, for example, byconventional northern blotting to quantitate the transcription of mRNA(Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980)), dot blotting(RNA analysis), or in situ hybridization, using an appropriately labeledprobe, based on the sequences that encode the polypeptide. Variouslabels may be employed, most commonly radioisotopes, particularly ³²P.However, other techniques may also be employed, such as usingbiotin-modified nucleotides for introduction into a polynucleotide. Thebiotin then serves as the site for binding to avidin or antibodies,which may be labeled with a wide variety of labels, such asradionuclides, fluorescers, enzymes, or the like. Alternatively, assaysor gels may be employed for detection of protein.

For secretion of an expressed gene product, the host cell is culturedunder conditions sufficient for secretion of the gene product. Suchconditions include, e.g., temperature, nutrient, and cell densityconditions that permit secretion by the cell. Moreover, such conditionsare those under which the cell can perform the basic cellular functionsof transcription, translation, and passage of proteins from one cellularcompartment to another, as are known to those skilled in the art.

-   F. Purification of Polypeptides

The following procedures, individually or in combination, are exemplaryof suitable purification procedures, with the specific method(s) usedbeing dependent on the type of polypeptide: fractionation onimmunoaffinity or ion-exchange columns; ethanol precipitation;reversed-phase HPLC; hydrophobic-interaction chromatography;chromatography on silica; chromatography on an ion-exchange resin suchas S-SEPHAROSE™ and DEAE; chromatofocusing; SDS-PAGE; ammonium-sulfateprecipitation; and gel filtration using, for example, SEPHADEX™ G-75medium.

The monoclonal antibodies may be suitably separated from the culturemedium by conventional antibody purification procedures such as, forexample, protein A-SEPHAROSE™ medium, hydroxyapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature and patent citations hereinare incorporated by reference.

EXAMPLE 1 Feeding of G3P to Shake Flask Culture for the Production ofLlama Antibody Fragment (Heavy Chain) and Apo2L

Background:

The inclusion of 200 mM G3P (final concentration) in eitherlow-phosphate (CRAP) or high-phosphate culture medium (THCD) wascompared to the respective control addition (water) for the expressionof a heterologous protein in shake-flask culture. In the first part ofthis Example, the target heterologous protein is a 13 kD llama anti-HCGcamelid monobody. Camelid antibodies have been previously shown to have2 species, a classic IgG molecule consisting of two heavy plus two lightchains and a heavy-chain IgG molecule lacking a light chain referred toas monobody. The camelid monobody was expressed by BL21, an E. coli Bstrain, using a tac promoter in either a low-phosphate (CRAP)- or ahigh-phosphate (THCD) -rich media. The malE binding protein signalsequence preceding the antibody-fragment-encoding sequence directed thesecretion of the expression protein into the periplasm of the host. Inthe second part of this Example, a T7 promoter was used to regulate theexpression of Apo2 ligand in HMS174, an E. coli K12 strain, inG3P-supplemented and unsupplemented CRAP medium. Production ofheterologous protein in both experiments was induced with the additionof IPTG upon reaching the desired cell density.

Materials & Methods:

pCB36624 86.RIG Plasmid Construction:

pCB36624_(—)86.RIG_was constructed by modifying vector pL1602 (Sidhu etal., J. Mol. Biol., 296:487-495 (2000)). Vector pS1602, which has pTacpromoter sequence and malE secretion signal sequence, contained asequence of human growth hormone fused to the C-terminal domain of thegene-3 minor coat protein (p3) of phage mu. The sequence encoding hGHwas removed and the resulting vector sequence served as the vectorbackbone for the insertion of a synthetic DNA fragment encoding thellama anti-HCG antibody (Spinelli et al., Nat. Struct. Biol. 3(9):752-757 (1996)). The resulting phagemid (pCB36624) encoded the fusionproduct under the control of the IPTG-inducible P_(tac) promoter (Ammanand Brosius, Gene, 40: 183-190 (1985)). The expressed polypeptideincluded the maltose-binding protein signal peptide, followed by theanti-HCG coding region, followed by a FLAG epitope tag, followed by aGly/Ser-rich linker peptide containing a suppressible stop codon,followed by P3C (the C-terminal domain of the phage coat protein).

Phage-displayed libraries were constructed using the method of Sidhu etal., J. Mol. Biol., 296: 487-495 (2000) with appropriately designed“stop template” phagemids. For library NNS17, a derivative of pCB36624that contained TAA stop codons in place of codons 93, 94, 100 and 101was used as the template for the Kunkel mutagenesis method (Kunkel etal., Methods Enzymol., 154: 367-382 (1987)), with mutagenicoligonucleotide NNS17 designed to simultaneously repair the stop codonsand introduce 17 NNK degenerate codons between the codons encoding Gly95and Trp103. NNS17: (SEQ ID NO:3) GCC GTC TAT ACT TGT GGT GCT GGT NNS NNSNNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS NNS TGG GGT CAGGGT

Like all monobodies, the llama anti-HCG is a Vh3 family member and assuch is recognized by Protein A. The Protein A binding interaction wasused as a surrogate for CDR3-mediated stability. The resulting phagelibraries were sorted by multiple rounds against Protein A as readout ofscaffold stability and expression. The sorted libraries were analyzedfor selection bias in the distribution of amino acids in the NNSlibrary. Scaffold RIG, as named by the sequence at positions 96, 97 and98, turned out to be the most dominant clone based on the sequencedresidues. The 17-amino-acid-long CDR3 sequence for Scaffold RIG wasdetermined to be RIGRSVFNLRRESWVTW (SEQ ID NO:4). The phagemid withScaffold RIG is renamed pCB36624_(—)86.RIG, with the DNA sequence:5′-GATGTTCAGT TGCAGGAATC AGGCGGTGGC (SEQ ID NO:5) TTGGTACAGG CCGGAGGTTCGTTGCGTTTG TCCTGTGCTG CCTCGGGTGC TACTGGTTCT ACTTATGATA TGGGCTGGTTTCGTCAGGCT CCGGGTAAAG AACGTGAATC GGTTGCCGCC ATTAACTGGG GGTCGGCTGGGACTTACTAT GCTTCGTCCG TCCGTGGTCG TTTTACTATT TCACGTGATA ATGCCAAAAAAACTGTCTAT TTGCAGATGA ATTCATTGAA ACCAGAAGAT ACTGCCGTCT ATACTTGTGGTGCTGGTAGG ATCGGCCGGT CGGTCTTCAA CTTGAGGAGG GAGAGCTGGG TCACGTGGTGGGGTCAGGGT ACCCAGGTCA CTGTCTCCTC TGCCGGTGGT ATGGATTATA AAGATGATGATGATAAA-3′pet19b.nohis Plasmid Construction

Using standard molecular biology techniques, Apo2L codons 114-281 wereamplified by polymerase chain reaction from a full-length Apo2L cloneisolated from human placental cDNA. Additional nucleotides containingrestriction sites to facilitate cloning are added to the 5′ and 3′sequences, respectively. The 5′ oligonucleotide primer has the sequence:(SEQ ID NO:6) 5′ GCTTGCTACATATGGTGAGAGAAAGAGGTCCTCAGAGA 3′

containing the underlined Nde I restriction site. The 3′ oligonucleotideprimer has the sequence: (SEQ ID NO:7)5′ CTTGAATAGGATCCCTATTAGCCAACTAAAAAGGCCCCAAAAAAACT GGC 3′containing the underlined BamH I restriction site. The resultingfragment was subcloned using the restriction sites Nde I to BamH I intoa modified baculovirus expression vector pVL1392 (Pharmingen) in frameand downstream of a sequence containing a His10 tag and an enterokinasecleavage site (Pitti et al., J. Biol. Chem., 271:12687-12690 (1997)).pVL1392-Apo2L was digested with Nde I and BamH I and the Nde I-to-BamH Ifragment generated was subcloned into pET-19b (Novagen), also digestedwith Nde I and BamH I. The resultant plasmid was named pet19b.nohis.Bacterial Strains:

Competent cells of BL21 (Stratagene) and HMS174 (Merck) were transformedwith pCB36624_(—)86.RIG and pet19b.nohis, respectively, using standardprocedures. Transformants were picked after growth on an LB platecontaining 50 μg/mL carbenicillin (LB+CARB50™ carbenicillin),streak-purified, and grown in LB broth with 50 μg/mL CARB50™carbenicillin in a 30° C. incubator. pCB36624_(—)86.RIG conferredcarbenicillin resistance to the production host BL21/ pCB36624_(—)86.RIGand pet19b.nohis to HMS174/pet19b.nohis, allowing the transformed hoststo grow in the presence of the antibiotic.

Fermentation Medium:

Both low-phosphate (CRAP) culture medium and high-phosphate (THCD)culture medium were used for the evaluation of production of llamaantibody fragment and Apo2 ligand. The media composition (with thequantities of each component utilized per liter of initial medium) islisted below: Low-PO₄ Medium High-PO₄ Medium Ingredient Quantity/LQuantity/L Glucose 5.5 g 5.5 g Ammonium Sulfate 3.57 g 3.57 g Na₂HPO₄ —1.86 g NaH₂PO₄—H₂O — 0.93 g Sodium Citrate, Dihydrate 0.71 g 0.71 gPotassium Chloride 1.07 g 1.07 g 1M Magnesium Sulfate 7 ml 7 ml HycaseSF 5.36 g — Yeast Extract 5.36 g 5.36 g Casamino Acids — 5.36 g 1M MOPS,ph 7.3 110 ml 110 ml KOH for pH adjustment as needed as needed to pH 7.3

To prepare 200 mM of G3P-supplemented medium, 5 ml of 1 MDL-alpha-glycerophosphate (G3P) (Sigma Chem. Co.) was added to 20 ml oflow-PO₄ medium with 50 μg/ml of carbenicillin (low-PO₄ medium+CARB50™carbenicillin) or high-PO₄ medium with 50 μg/ml of carbencillin(high-PO₄ medium+CARB50™ carbenicillin) prior to inoculation. For theunsupplemented (control) medium, 5 ml of water was used in place of G3P.

Shake-Flask Fermentation:

Shake-flask fermentation was conducted in a 125-ml baffled flaskcontaining 25 ml of control or G3P-supplemented medium. An overnightculture of BL21/pCB36624_(—)86.RIG or HMS174/pet19b.nohis grown inLB+CARB50™ carbenicillin was back-diluted at approx.1:100 forinoculation into the control or G3P-supplemented media. Cultures wereincubated at 30° C on a shaker at 250 RPM and product expression wasinduced by the addition of 1 mM of IPTG when cell density reachedapproximately 50-60% of the potential cell growth supported by themedium. Cell pellets from 1 ml of broth culture, taken just before theaddition of the inducer and at approximately 24 hrs post-inoculation,were prepared and stored at −20° C.

Llama Antibody Fragment Accumulation Analyzed by PAGE and Densitometry:

Frozen (−20° C.) cell pellet prepared from 1 ml of culture sample wasthawed and resuspended in sufficient quantity of 10 mM TRIS, pH 7.6+1 mMEDTA, pH 8.0 (TE) to bring the cell suspension to 1 OD/25 μlconcentration. 25 μl of the TE-cell suspension was mixed with 25 μl of2X sample buffer containing beta-mercaptoethanol. The mixture was heatedat >95° C. for 5 mins before 10 μl (equivalent to 0.2 OD) was loaded perwell onto NU-PAGE™ precasted 10% Bis-Tris gel (Novex). Electrophoresiswas performed in MES buffer (2-(N-morpholino) ethanesulphonic acid indeionised water adjusted to the appropriate pH, such as with 1 N NaOH).The resolved gel was stained with COOMASSIE BLUE R250™ stain and thendestained. The band intensity of the 13-kD antibody fragment wasdetermined using Kodak DIGITAL SCIENCE ID™ imaging software afterscanning the wet gel with the Kodak imaging system.

Apo2 Ligand Accumulation Analyzed by Reversed-Phase HPLC:

Frozen (−20° C.) cell pellet prepared from 1 ml of culture sample wasresuspended in sufficient quantity of TE buffer to bring the cellsuspension to 1 OD/25 μl concentration. 20 μl of the cell suspension wasmixed into 480 μl of 6 M guanidine HCl, pH 9.0+100 mM dithiothreitol(DTT), and was allowed to incubate at room temperature for an hourbefore being centrifuged at 13,000 rpm for 15 mins to recover thesupernatant/extract. The extract was filtered through a MILLIPORE™spin-filter before 20 μl was loaded onto an HPLC column (PerSeptiveBiosystems POROS® R1/10 medium) for reverse-phase chromatography. TheHPLC separation was conducted at 80° C. with the mobile phases flowingat 1.0 ml/min and employed a gradient of 28% to 35% of acetonitrile with0.1% TFA over 20 minutes for the resolution of the Apo2L away from thecontaminating proteins. Peak detection was at 280 nm wavelength. Theamount of monomer present in samples was calculated using an averageresponse factor (mAU/μg) derived from the area under the peak associatedwith 5-20 μg of purified standards analyzed by the same method.

Results:

FIG. 1 shows that the antibody is expressed to higher levels in bothhigh-PO₄ (THCD) and low-PO₄ (CRAP) medium supplemented with 200 mM G3Pversus the control.

FIG. 2 shows that the Apo2L protein is expressed to higher levels inlow-PO₄ (CRAP) medium supplemented with 200 mM G3P versus the control.

EXAMPLE 2 Feeding of G3P to 10-L Fermentor Culture of Wild-type or(ΔglpTphoA- ugp+) Host for Production of IGF-I Regulated by AlkalinePhosphatase Promoter

Materials & Methods:

pBKIGF-2B Plasmid for Expression of IGF-I:

The plasmid pBKIGF-2, used for the expression of IGF-I herein, wasconstructed as detailed in U.S. Pat. No. 5,342,763. This plasmid wasconstructed from a basic backbone of pBR322. The transcriptional andtranslational sequences required for expression of the IGF-I gene in E.coli are provided by the alkaline phosphatase promoter and the trpShine-Dalgarno sequences. The lambda t_(o) transcriptional terminator issituated adjacent to the IGF-I termination codon. Secretion of theprotein from the cytoplasm is directed by the lamB signal sequence. Themajority of rhIGF-I is found in the cell periplasmic space. PlasmidpBKIGF-2B confers tetracycline resistance upon the transformed host.

Bacterial Strains and Growth Conditions:

The hosts used in the IGF-I fermentation are derivatives of E. coliW3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington,D.C.: American Society for Microbiology, 1987), pp. 1190-1219).Experiments concerning a host with wild-type glpT were carried out withstrain 43E7 (E. coli W3110fhuA(tonA) A(argF-lac) ptr3 degP41AompTA(nmpc-fepE) ilvG+phoA), and experiments concerning a host with aΔglpT mutation were carried out with strain 43F6 (E. coliW3110fhuA(tonA) A(argF-lac) ptr3 degP41 ΔompTA(nmpc-fepE) ilvG+phoAΔglp)). Competent cells of 43E7 or 43F6 were transformed with pBKIGF-2Busing standard procedures. Transformants were picked after growth on anLB plate containing 20μg/mL tetracycline (LB +TET20TM tetracycline),streak-purified, and grown in LB broth with 20 μg/mL TET20™ tetracyclinein a 37° C. shaker/incubator before being tested in the fermentor.pBKIGF-2B confers tetracycline resistance to the production host andallows the transformed host to grow in the presence of the antibiotic.

10-L Fermentation Process:

The fermentation medium composition and run protocol used for theexpression of IGF-I were somewhat similar to those used in the IGF-Iprocess described in U.S. Pat. No. 5,342,763. Briefly, a shake-flaskseed culture of 43E7/pBKIGF-2 or 43F6/pBKIGF-2 was used to inoculate therich production medium. The composition of the medium (with thequantities of each component utilized per liter of initial medium) isdescribed below: Ingredient Quantity/L Glucose* 200-500 g AmmoniumSulfate 2-10 g Sodium Phosphate, Monobasic Dihydrate 1-5 g PotassiumPhosphate, Dibasic 1-5 g Sodium Citrate, Dihydrate 0.5-5 g PotassiumChloride 0.5-5 g Magnesium Sulfate, Heptahydrate 0.5-5 g PLURONIC ™Polyol, L61 0.1-5 mL Ferric Chloride, Heptahydrate 10-100 mg ZincSulfate, Heptahydrate 0.1-10 mg Cobalt Chloride, Hexahydrate 0.1-10 mgSodium Molybdate, Dihydrate 0.1-10 mg Cupric Sulfate, Pentahydrate0.1-10 mg Boric Acid 0.1-10 mg Manganese Sulfate, Monohydrate 0.1-10 mgHydrochloric Acid 10-100 mg Tetracycline 4-30 mg Yeast Extract* 5-25 gNZ Amine AS* 5-25 g Methionine* 0-5 g Ammonium Hydroxide as required tocontrol pH Sulfuric Acid as required to control pH*A portion of the glucose, yeast extract, methionine, and NZ Amine AS isadded to the medium initially, with the remainder being fed throughoutthe fermentation.

The 10-liter fermentation was a fed batch process with fermentationparameters set as follows: Agitation: 1000 RPM Aeration: 10.0 slpm pHcontrol: 7.3 Temp.: 37° C. Back pressure: 0.3 bar Glucose feed:computer-controlled using an algorithm to maintain the dissolved oxygenconcentration (DO₂) at 30% of air saturation after the DO₂ drops to 30%.Complex nitrogen feed: constant feed rate of 0.2 mL/min beginning whenOD₅₅₀ reaches 40 and maintained for the remaining time of the run RunDuration: 40 to 50 hours

In experiments involving glycerol-3-phosphate (G3P) feeding, theappropriate amount of 1 M G3P stock solution was spiked into the complexnitrogen feed and the subsequent supplemented feed feed-rate increasedto deliver the desired amount of complex nitrogen plus G3P to theculture.

The impact of the ΔglpT mutation with or without the G3P feeding wasassessed by the difference in the IGF-I accumulation. The total amountof IGF-I in a sample solubilized in 6M guanidine+100 mM DTT was measuredby a reversed-phase HPLC method as described in U.S. Pat. No. 6,559,122.

Results:

FIG. 3 shows that with the wild-type host (43E7) and AP promoter andcontinuously fed glucose, the amount of secreted IGF-I was distinctlyhigher when G3P was fed to the medium than when G3P was not added.

FIG. 4 shows that with the ΔglpT host (43F6) and AP promoter, the amountof secreted IGF-I was distinctly higher when G3P was fed to the cultureat 1.18 or 3.28 mmoles/hour, per approximately 8 liters, than when G3Pwas not added, but was not higher when 8.22 mmoles/hour, perapproximately 8 liters, of G3P was fed. The optimum feed rate will bereadily determined by one skilled in the art based on the product, typeof organophosphate, etc. Under the conditions of the fermentationprocess described, culturing in a 10-liter fermentor to produce IGF-I,there is an optimal G3P feed rate, per approximately 8-10 liters, in thepreferred range of about 1-7 mmoles/hour, more preferably about 1-6mmoles/hour, still more preferably about 2-6 mmoles/hour, yet morepreferably about 2-5 mmoles/hour, and most preferably about 3-4mmoles/hour. Not only does this range of feed rates increase the amountof product over control, but also it extends the duration of productionrelative to the control.

EXAMPLE 3 Feeding of Glycero-3-phosphate to Improve Apo2 LigandAccumulation in the 10-L Process

Background on Apo2 Ligand

Apoptosis-inducing ligand 2 (Apo2L) (Pitti et al., J. Biol. Chem., 271:12687-12690 (1996)), also known as tumor necrosis factor-relatedapoptosis inducing ligand (TRAIL) (Wiley et al., Immunity, 3: 673-682(1995)), is a type II membrane protein and a member of the TNF family ofligands. Apo2L/TRAIL triggers apoptosis in a wide variety of cancercells, but not in most normal cells, through binding to its cognatedeath receptors (WO 99/00423; Ashkenazi, FASEB J., 13: (7) A1336 (Apr.23, 1999); Ashkenazi, Nature Reviews—Cancer, 2: 420-430 (2002)). Asoluble fragment of the extracellular domain of Apo2 ligand,corresponding to amino acid residues 114-281 (from here on referred toas Apo2L/TRAIL), is currently under investigation for potential clinicalstudies and has been successfully expressed in E. coli.

General Description of the Fermentation Process:

The expression vector encodes for the use of the alkaline phosphatase(AP) promoter to regulate the production of the approximately 19.5-kDapolypeptide. The expressed nascent polypeptides, upon release from theribosomes, fold into monomers in the cytoplasm and further associate tobecome the biologically active homotrimer. During fermentation, theprocess parameters are set such that cellular activities are conductedat peak oxygen uptake rates of approximately 3.0 mmoles/L-min. Afterbroth harvest, the cytoplasmically trapped heterologous protein isreleased by mechanical cell disruption into the cell lysate from whichit may be recovered.

Materials and Methods:

pAPApo2-P2RU Plasmid Construction:

pAPApo2-P2RU is described in WO 01/00832 published Jan. 4, 2001.Briefly, this plasmid, the construct of which is shown in FIG. 5,encodes the co-expression of Apo-2L (amino acid residues 114-281) andthe rare-codon tRNA's encoded by pro2 and argU, which co-expression isregulated by the alkaline phosphatase promoter. The pBR322-based plasmid(Sutcliffe, Cold Spring Harbor Symp. Quant. Biol., 43:77-90 (1978))pAPApo2-P2RU was used to produce the Apo-2L in E. coli. Thetranscriptional and translational sequences required for the expressionof Apo-2L are provided by the alkaline phosphatase promoter and the trpShine-Dalgarno sequence, as described for the plasmid phGHI (Chang etal., Gene, 55:189-196 (1987)). The coding sequence for Apo-2L (from114-281) is located downstream of the promoter and Shine-Dalgarnosequences and is preceded by an initiation methionine. The codingsequence includes nucleotides (shown in FIG. 6) encoding residues114-281 of Apo-2L (FIG. 6—SEQ ID NOS:1 and 2, respectively, fornucleotide and amino acid sequences) except that the codon encodingresidue Pro119 is changed to “CCG” instead of “CCT” in order toeliminate potential secondary structure. The sequence encoding thelambda to transcriptional terminator (Scholtissek et al., Nucleic AcidsRes., 15: 3185 (1987)) follows the Apo-2L coding sequence.

Additionally, this plasmid also includes sequences for the expression ofthe tRNA's pro2 (Komine et al., J. Mol. Biol., 212:579-598 (1990)) andargU/dnaY (Garcia et al., Cell, 45:453-459 (1986)). These genes werecloned by PCR from E. coli W31 10 and placed downstream of the lambda totranscriptional-terminator sequence. This plasmid confers bothtetracycline and ampicillin resistance upon the production host.

Bacterial Strains and Growth Conditions:

Strain 43E7 (E. coli W3110 fhuA(tonA) phoa Δ(argF-lac) ptr3 degP ompTilvG+)) was used as the wild-type production host for comparison to43F6, the glpT-mutated host for the expression of Apo2 ligand and therare codon tRNA's. Competent cells of 43E7 or 43F6 were prepared andtransformed with pAPApo2-P2RU using standard procedures. Transformantswere picked from LB plates containing 20 μg/ml tetracycline (LB+Tet20),streak-purified, and grown in LB broth with 20 μg/ml tetracycline in a30° C. shaker/incubator before being stored in DMSO at −80° C.

Fermentation Process for Apo2L Production:

A shake-flask inoculum was prepared by inoculating sterile LB mediumcontaining 4-6 mM sodium phosphate with a freshly thawed stock culturevial. Appropriate antibiotics were included in the medium to provideselective pressure to ensure retention of the plasmid. Flask cultureswere incubated with shaking at about 30° C. (28° C.-32° C.) for 14-18hours. This culture was then used to inoculate the productionfermentation vessel. The inoculation volume was between 0.1% and 10% ofthe initial volume of medium.

Production of Apo2L was carried out in the production medium given inTable 1 to achieve a final culture volume of approximately 10 liters.The fermentation process was conducted at about 30° C. (28-32° C.) andpH controlled at approximately 7.0 (6.5-7.5). The aeration rate and theagitation rate were set to provide adequate transfer of oxygen to theculture. Just prior to depletion of the batched phosphate (atapproximately 75-85 OD), a DL-alpha-glycerophosphate feed (vendorproduct specification shows product purity at 80-90%, withbeta-glycerophosphate listed as the main impurity) was initiated and fedat the desired feed rate. Throughout the fermentation process, the cellculture was fed glucose as the primary carbon source based on a computeralgorithm while ensuring aerobic conditions.

Two batch additions of approximately 50-150 μM (final concentration)ZnSO₄ were made during the fermentation process, one just prior to theinduction of product expression, the other at approximately themid-point of the production period for improved homotrimer assembly. Inthis example, the additions occurred at a culture optical density ofabout 80-120 OD₅₅₀ and at about 28 hours post-inoculation.

The fermentation was allowed to proceed for about 34-45 hours beforebeing harvested. TABLE 1 Production Medium Composition for AP PromoterExpression System Ingredient Quantity/Liter Tetracycline 4-20 mgGlucose^(a) 10-250 g Ammonium sulfate^(a) 2-8 g Sodium phosphate,monobasic, dihydrate^(a) 1-5 g Potassium phosphate, dibasic^(a) 1-5 gPotassium phosphate, monobasic^(a) 0-5 g Sodium citrate, dihydrate^(a)0.5-5 g Potassium chloride 0-5 g Magnesium sulfate, heptahydrate^(a)1.0-10 g Antifoam 0-5 ml Ferric chloride, hexahydrate^(a) 20-200 mg Zincsulfate, heptahydrate^(a) 0.2-20 mg Cobalt chloride, hexahydrate^(a)0.2-20 mg Sodium molybdate, dihydrate^(a) 0.2-20 mg Cupric sulfate,pentahydrate^(a) 0.2-20 mg Boric acid^(a) 0.2-20 mg Manganese sulfate,monohydrate^(a) 0.2-20 mg Casein hydrolysate^(a) 5-25 g Yeastextract^(a) 5-25 g^(a)A portion of these ingredients may be fed to the culture during thefermentation. Ammonium hydroxide was added as required to control pH.Assessment of Soluble Product Accumulation During Fermentation Processby Ion-Exchange HPLC Chromatography Method:

Broth samples were taken over the time course of the fermentationprocess. Cells from 1 milliliter of broth samples diluted to a celldensity of 20 OD₅₅₀ were collected by centrifugation and the resultantcell pellets were stored at −20° C. until analysis. The cell pelletswere thawed and resuspended in 0.5 ml of extraction buffer (50mM HEPES,pH 8.0, 50 mM EDTA and 0.2 mg/ml hen egg-white lysozyme) andmechanically disrupted to release the product from the cytoplasm. Solidswere removed from the cell lysates by centrifugation before theclarified lysates were loaded onto an HPLC column (DIONEX PROPAC™ IEXmedium) for trimer quantitation. The HPLC assay method resolved theproduct away from the contaminating E. coli proteins by use of a 5%-22%gradient of 1M NaCl in a 25-mM phosphate (pH 7.5) buffer over 25 minutesat a flow rate of 0.5 ml/min.

Assessment of Total Monomeric Apo2L Expression During FermentationProcess by Reversed-Phase HPLC Chromatography:

Fresh culture broth or previously frozen and then thawed samples wereused for the quantitation of total monomer production. 20 μl of samplewas mixed into 480 μl of 6M guanidine HCl, pH 9.0 with 100 mM DTT andwas allowed to incubate at room temperature for an hour before beingcentrifuged at 13,000 rpm for 15 mins to recover the extract. Theextract was filtered through a spin-filter before 20 μl was loaded ontoan HPLC column (PerSeptive Biosystems POROS® R1/10 medium) forreverse-phase chromatography. The HPLC separation was conducted at 80°C. with the mobile phases flowing at 1.0 ml/min and employed a gradientof 28% to 35% of acetonitrile with 0.1% TFA over 20 minutes for theresolution of the Apo2L away from the contaminating proteins. Peakdetection was at 280-nm wavelength. The amount of monomer present insamples was calculated using an average response factor (mAU/μg) derivedfrom the area under the peak associated with 5-20 μg of purifiedstandards analyzed by the same method.

Results:

FIG. 7 shows an improved specific product titer (referred to as specifictiter in μg/OD-ml in the graph) with an optimum G3P feed rate to theΔglpT host (43F6). All of the G3P-fed runs performed better than theno-feed control. In this example, as the feed rate for an approximately8-liter culture increased from 6 to 12 mmole/hour, the specific producttiter improved, but as the rate increased above 12 mmole/hour to 18mmole/hour, the specific titer was lower. The optimum feed rate of G3Pwill be readily determined by one skilled in the art based on theproduct, type of organophosphate, etc. Under these particularconditions, culturing in a 10-liter fermentor cells for producing thisspecific product, Apo2L, the preferred feed rate of G3P, perapproximately 8-10 liters, is preferably in the range of about 4 to 17mmole/hour, more preferably about 6 to 16 mmole/hour, still morepreferably about 8 to 15 mmole/hour, and most preferably about 10 to 14mmole/hour.

FIG. 8 shows an improved specific product titer (referred to as specifictotal accumulation in μg/OD-ml in the graph) feeding G3P over feedinginorganic phosphate to the wild-type glpT host (43E7). Whileglycerophosphate feeding increased specific total accumulation of Apo2L,feeding inorganic phosphate negatively impacted specific totalaccumulation compared to the no-feed control. Similar trends would beexpected using a lower glycerophosphate feed than was employed. Theresults here are intended to, and do, show that a high level ofexpression can be obtained by feeding glycerophosphate to a wild-typegipT host. Further, in this particular experiment, similar to theinorganic phosphate feed case, the culture cell density increased toover 200 OD550 when the glycerophosphate was fed, but not for theno-feed situation.

EXAMPLE 4 Expression of AP Promoter-Driven Apo2L Product during ActiveGrowth Phase

The same plasmid construction, production host strain, mediumcomposition, fermentation process and product assay methods were used asdescribed in Example 3 except for the phosphate batching and the G3Paddition. A portion of the inorganic phosphate typically included in thesalt batching in a control process was replaced with an equivalentnumber of moles of G3P, either added immediately after inoculation or afew hours prior to the depletion of the batched inorganic phosphate. Inthese examples, the added G3P was expected to be the source of phosphatefor a significant fraction of the cell growth subsequent to theaddition.

Fermentation Process for Apo2L Production during Active Growth Phase:

The inoculum preparation protocol was the same as that described inExample 3. Production of Apo2L was carried out in the production mediumgiven in Table 1 except that either 75% or 50% of the phosphate saltswas eliminated from the initial batching and replaced with an equivalentnumber of moles of G3P added back as a batch addition post inoculation.The fermentation was conducted at about 30° C. (28-32° C.) and pH wascontrolled at approximately 7.0 (6.5-7.5) as per standard protocol. Theaeration rate and the agitation rate were as described in Example 3. Forthe case where 50% of the inorganic phosphate was replaced with G3P, theinorganic phosphate was hatched in prior to medium sterilization whilethe glycerol-3-phosphate replacement was made approximately 1-2 hoursbefore the hatched phosphate was expected to run out (at approximately30-40 OD₅₅₀). For the case where 75% of the inorganic phosphate wasreplaced with G3P, both the inorganic phosphate and the G3P were addedimmediately after the inoculation of the fermentor. Throughout thefermentation process, the cell culture was fed glucose as the primarycarbon source based on a computer algorithm while ensuring aerobicconditions. Zn additions were made during the fermentation process asdescribed in the earlier section. The fermentation was allowed toproceed for about 34-45 hours.

Results:

FIG. 9 shows the induction of heterologous protein expression occurringsignificantly earlier in the active growth phase when 50%-75% of the PO₄batching was replaced with G3P addition for both the wild-type andgipT-mutated hosts, shifting the specific total accumulation curve tothe left of that for the duplicate control cases conducted with thewild-type host with no G3P substitution. This indicates an advantage ofthis invention in that the product can be obtained earlier during thefermentation process.

While all ratios of Pi to G3P tested herein achieved this advantageregardless of the host type, Table 2 shows that using the 1:1 or 1:3ratio of Pi to G3P for the glpT-mutated host 43F6 produced the highestvolumetric Apo2L productivity rate (an average of about 0.34 versus anaverage of about 0.24 mg/ml-hr for the control host). Further, usingeither ratio and the wild-type or mutated host achieved the peakspecific accumulation (in μg/OD-ml) earlier (22 to 26 hours versus 28 to30 hours). This shows that in certain preferred embodiments, theinvention can achieve similar, if not higher, amounts of monomeric Apo2Lin approximately 10% to 25% less fermentation time than otherwise toimprove process productivity significantly. TABLE 2 Effect of ReplacingInorganic Phosphate Initial Batching with Glycerophosphate AdditionDuring the First 30 Hours of Fermentation Volumetric Time to Peak PeakTotal Productivity Specific monomeric Rate Accum. Apo2L Yield Experiment(mg/ml-hr) (μg/OD-ml) (g/L) Control (43E7) 0.27 28 2.9 Control (43E7)0.21 30 2.8 Pi/G3P @ 1:1 (43F6) 0.34 22.5 3.3 (50% replacement) Pi/G3P @1:1 (43E7) 0.25 22 2.0 (50% replacement) Pi/G3P @ 1:3 (43F6) 0.34 26.03.0 (75% replacement)

EXAMPLE 5 Expression of AP Promoter-Driven Apo2L Product Using a 50/50Mixture of Alpha- and Beta- Glycerophosphate

A procedure similar to that described in Example 3 was followed exceptthat a cheaper grade of approximately 50:50 mix of alpha- andbeta-glycerophosphate was employed instead of G3P as the feed usingstrain 61G1 (glpT mutant host).

Results

FIG. 10 shows that similar yield improvement over the no-feed controlwas obtained using the mixture or the higher grade G3P material. Use ofthe alpha/beta mixture would lessen the cost of raw material withoutcompromising the production results.

1. A process for producing a polypeptide heterologous to E. colicomprising (a) culturing E. coli cells comprising nucleic acid encodingthe polypeptide in a culture medium while feeding to the culture mediuma transportable organophosphate, such that the nucleic acid isexpressed, and (b) recovering the polypeptide from the cells.
 2. Theprocess of claim 1 wherein the organophosphate is a glycerophosphate. 3.The process of claim 2 wherein the glycerophosphate is analpha-glycerophosphate or beta-glycerophosphate, or a mixture thereof.4. The process of claim 3 wherein the glycerophosphate is a mixture ofglycerol-2-phosphate and glycerol-3-phosphate or isglycerol-3-phosphate.
 5. The process of claim 1 wherein the culturingtakes place in a shake flask or fermentor.
 6. The process of claim 1wherein the polypeptide is recovered from the cytoplasm, periplasm orculture medium of the cells.
 7. The process of claim 1 whereinexpression of the nucleic acid is regulated by an inducible promoter. 8.The process of claim 7 wherein the inducible promoter is the alkalinephosphatase promoter.
 9. The process of claim 7 wherein the induciblepromoter is the tac promoter.
 10. The process of claim 7 wherein theinducible promoter is the T7 promoter.
 11. The process of claim 7wherein expression of the nucleic acid begins while in the active growthphase of the culturing step.
 12. The process of claim 1 wherein the E.coli is deficient in chromosomal phoA.
 13. The process of claim 1wherein the E. coli is wild type with respect to chromosomal gipT. 14.The process of claim 1 wherein the E. coli is deficient in chromosomalglpT.
 15. The process of claim 1 wherein the E. coli is deficient inchromosomal phoA and glpT.
 16. The process of claim 15 wherein the E.coli is not deficient in chromosomal ugp.
 17. The process of claim 1wherein the polypeptide is a eukaryotic polypeptide.
 18. The process ofclaim 1 wherein the polypeptide is a mammalian polypeptide.
 19. Theprocess of claim 1 wherein the polypeptide is insulin-like growthfactor-1.
 20. The process of claim 19 wherein the feed rate of theorganophosphate is from about 1 to 7 mmoles/hour per about 8-10 litersand the culturing takes place in a 10-liter fermentor.
 21. The processof claim 20 wherein the feed rate of the organophosphate is from about 2to 6 mmoles/hour per about 8-10 liters.
 22. The process of claim 21wherein the feed rate of the organophosphate is from about 3 to 4mmoles/hour per about 8- 10 liters.
 23. The process of claim 1 whereinthe polypeptide is Apo2L.
 24. The process of claim 23 wherein the feedrate of the organophosphate is from about 4 to 17 mmoles/hour per about8-10 liters and the culturing takes place in a 10-liter fermentor. 25.The process of claim 24 wherein the feed rate is from about 6 to 16mmole/hour per about 8-10 liters.
 26. The process of claim 25 whereinthe feed rate is from about 8 to 15 mmole/hour per about 8-10 liters.27. The process of claim 26 wherein the feed rate is from about 10 to 14mmole/hour per about 8-10 liters.
 28. The process of claim 1 wherein aninorganic phosphate is also present during the culturing step.
 29. Theprocess of claim 28 wherein the ratio of inorganic phosphate toorganophosphate ranges from about 1:10 to 1:0.25.
 30. The process ofclaim 29 wherein the polypeptide is Apo2L and the ratio is about 1:3 to1:0.5.
 31. The process of claim 30 wherein the ratio is about 1:1.